Guanylate binding proteins (GBP) were first identified as interferon-inducible proteins in fibroblasts (Cheng et al., (1983) J. Biol. Chem. 258:7746–7750). They were identified by their ability to bind to guanylate-containing agaroses including GMP-agarose, GDP-agarose, and GTP-agarose. They failed to bind to ATP-agarose and all other nucleotide agaroses tested (Cheng et al., (1991) Mol. Cell. Biol. 11:4717–4725). Purified GBP-1 protein was shown to hydrolyze GTP, but not ATP, UTP, or CTP with a Km for substrate of 470 μM. The hydrolysis reaction occurred with a high turnover rate and yielded primarily GMP rather than GDP (Schwemmle et al., (1994) J. Biol. Chem. 269:11299–11305). The hydrolysis reaction was thought to occur in two successive steps since pyrophosphate was not detected as a reaction product. GBP-1 was also shown to possess a functional CX1X2X3 (SEQ ID NO:1) isoprenylation signal at the C terminus of the molecule (the signal comprises a cysteine residue (C) followed by two typically, but not necessarily, aliphatic amino acids (X1 and X2) and another amino acid (X3)).
The crystal structure of human GBP-1 in nucleotide free (Prakash et al., (2000) Nature 403:567–571) and bound states (Prakash et al., (2000) EMBO J. 17:4555–4564) was solved to 1.8 Å resolution. The structure resembled that of other large GTP-binding proteins including Mx and dynamin. The structure consists of a compact amino-terminal globular α,β domain and an elongated C-terminal α-helical domain separated by a short intermediate region. The globular domain contains the conserved regions characteristic of GTP-binding proteins with some additional insertions. Significant differences in the glycosidic bond angle and guanine base interaction domains were observed in GBP-1 relative to the canonical GTP binding protein Ras. Unlike Ras, the phosphate binding region of GBP1 is closed off from solvent, and therefore unavailable for interactions with external regulators such as GTPase activating proteins or guanine nucleotide exchange proteins. Similar to dynamins, nucleotide binding to GBP-1 induced oligomerization.
Expression of GBP-1 is regulated by interferon in fibroblasts and a number of other cell types. These include mouse 70Z/3 pre-B cells (Patrone et al., (2001) Mol. Immunol. 38:597–606); murine macrophage lines and peritoneal macrophages (Wynn et al., (1991) J. Immunol. 147:4384–4392); rat bone marrow-derived macrophages and microglia (Vestal et al., (1996) Bioch. Biophys. Res. Commun. 224:528–534); cultured mammary epithelial tumor cell lines (Sun et al., (1999) Int. J. Cancer 80:624–629); normal and transformed keratinocytes (Saunders et al., (1999) J. Invest. Dermatol. 112:977–983); and human endothelial cells (Lubeseder-Martellato et al., (2002) Am. J. Pathol. 161:1749–1759). Lipopolysaccharide (LPS) was also shown to induce GBP-1 in rat bone marrow derived macrophages and microglia (Vestal et al., (1996) Bioch. Biophys. Res. Commun. 224:528–534), and in cultured mammary epithelial tumor lines (Sun et al., (1999) Int. J. Cancer 80:624–629). TNFα stimulation of human endothelial cells also induced GBP-1 expression (Lubeseder-Martellato et al., (2002) Am. J. Pathol. 161:1749–1759). In this study, GBP-1 expression was highly associated with vascular endothelial cells and was induced in vessels of skin diseases that have a high inflammatory component.
Since the original characterization of GBP-1, a number of related proteins have been identified in both mouse and human. In human, known GBPs include GBP-1, GBP-2 (Cheng et al., (1991) Mol. Cell. Biol. 11:4717–4725), GBP-4, and GBP-5. GBP-2 was shown to have similar nucleotide binding properties and GTPase activities as GBP-1 (Neun et al., (1996) FEBS Lett. 390:69–72). Some differences were noted in the product specificity of the GTPase reaction.
In mouse, the known GBPs include mGBP-1, mGBP-2 (Boehm et al., (1998) J. Immunol. 161:6715–6723), mGBP-3 (Han et al., (1998) Bioch. Biophys. Acta 1384:373–386), mGBP-4/mag-2 (Wynn et al., (1991) J. Immunol. 12:4384–4392) and mGBP-5 (Nguyen et al., (2002) J. Interferon Cytokine Res. 22:899–909). Recombinant mGBP-3 protein was shown to possess similar nucleotide binding properties and GTPase activity as compared to other GBPs (Han et al., (1998) Bioch. Biophys. Acta 1384:373–386). The tissue expression and induction patterns of all the mouse GBPs were similar (Nguyen et al., (2002) J. Interferon Cytokine Res. 22:899–909). All five were induced in multiple organs in response to endotoxemia. In RAW264 and Swiss 3T3 cells, all five GBPs were induced in response to LPS, interleukin-1β (IL-1β), and TNFα.
Although GBP-1 represents one of the most abundant proteins induced in response to interferon treatment, its function, or the function of other family members is not clear. Stable transfection of human GBP-1 into HeLa cells increased the resistance of the cells to the cytopathic effects of both vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV, Anderson et al., (1999) Virology 256:8–14). Conversely, cells transfected with GBP-1-specific antisense were more sensitive to viral infection, suggesting that GBP-1 is required for an antiviral response to VSV and EMCV viruses.
Expression of GBP-1 was inversely correlated with the proliferation of human microvascular and macrovascular endothelial cells (Guenzi et al., (2001) EMBO J. 20:5568–5577). GBP-1 expression is induced by pro-inflammatory, anti-proliferative cytokines including IL-1β, TNFα, and TFN-γ. Overexpression of full length GBP-1 in HUVECs significantly slowed the proliferative rate of these cells without increasing cell death. Conversely, expression of antisense to GBP-1 in HUVECs resulted in increased proliferation of the cells. Overexpression of GBP-1 also inhibited the proliferative response of the cells to VEGF and bFGF. Activation of the MAP kinase pathway in response to VEGF or bFGF was not affected by GBP-1 overexpression. Overexpression did not inhibit IL-1β-induced adhesion of the cells to monocytes. The anti-proliferative effect of GBP-1 was mapped to the C-terminal α-helical domain. Mutants lacking GTPase activity or the prenylation motif were still able to inhibit proliferation. Expression of the α-helical domain alone inhibited proliferation whereas expression of the globular nucleotide binding domain alone was inactive.
In contrast to the anti-proliferative effect of GBP1 in HUVECs, overexpression of mGBP-2 in NIH3T3 stimulated proliferation (Gorbacheva et al., (2002) J. Biol. Chem. 277:6080–6087). Cells constitutively expressing mGBP-2 formed foci when grown to post-confluence. They failed to grow in soft agar suggesting that they retained anchorage-dependent growth. These cells also grew as tumors in nude mice. Mutation of the nucleotide binding domain of mGBP-2 eliminated the effect on cell proliferation. This study suggests that mGBP-2 is able to alter the growth characteristics of fibroblasts.